Cells were homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), 0.1% Sodium dodecyl sulfate (SDS), 1% sodiumdeoxycholate and protease and phosphatase inhibitor cocktails (Sigma, Schnelldorf, Germany). After sonication and centrifugation, protein concentration was measured with the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). A total of 20 µg of protein were loaded on an SDS-PAGE gel and transferred to a nitrocellulose membrane. Following blocking, filters were incubated with antibodies directed against His, MBP, phosphor–AMPK (Thr172), alpha–AMPK, phospho–AS160 (Thr642), phospho–Akt (Thr308), Akt, ACC, phospho–ACC (all Cell Signaling Technology, Europe B.V., Frankfurt am Main, Germany), GLUT1, GLUT4 (both from Cushman, S.W. [32 (link)]), MBP (NEB), AS160 (Merck Millipore, Darmstadt, Germany) and GAPDH (Abcam, Cambridge, UK). After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab, Erlangen, Germany).
Ampk alpha
Manufactured by Cell Signaling Technology
Sourced in United States
AMPK alpha is a catalytic subunit of the AMP-activated protein kinase (AMPK) enzyme complex. AMPK alpha is responsible for the phosphorylation and activation of the AMPK enzyme, which plays a crucial role in the regulation of cellular energy homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Fusion fx7 imaging system, by Avantor (3 mentions)
Phospho ampk alpha thr172, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Cox 1, by Thermo Fisher Scientific (2 mentions)
Phospho ampk, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Phosphoampk alpha, by Cell Signaling Technology (2 mentions)
Si con, by Thermo Fisher Scientific (2 mentions)
Am17110, by Thermo Fisher Scientific (2 mentions)
Am17010, by Thermo Fisher Scientific (2 mentions)
Sc 271389, by Proteintech (2 mentions)
Mouse preimmune iggs, by Merck Group (2 mentions)
Siksrp, by Thermo Fisher Scientific (2 mentions)
Sidnmt3b, by Santa Cruz Biotechnology (2 mentions)
Sih19, by Thermo Fisher Scientific (2 mentions)
Am17100 pm10050, by Thermo Fisher Scientific (2 mentions)
13 protocols using ampk alpha
1
Protein Extraction and Western Blotting
2
Isolation and Analysis of Cellular Signaling Proteins
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Cells and tissues were homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate and protease and phosphatase inhibitor cocktails (Sigma) to isolate whole cell lysates. Isolation of the membrane fraction was performed with the Mem-PERTM Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). After sonication and centrifugation, protein concentration was measured with the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). 50 μg of protein were loaded on a SDS-PAGE gel and transferred to a nitrocellulose membrane. Following blocking, filters were incubated with antibodies directed against, phospho-AMPK (Thr172), alpha-AMPK, phospho-Akt (Thr308), total-Akt, phospho-AS160 (Thr642), total-AS160 (all Cell Signaling Technology), PCSK9 (Abcam), ND1 (Abcam), Cox I (Thermo Fisher Scientific), Na-K-ATPase and GAPDH (both Abcam). In order to rule out unspecific binding, the PCSK9 antibody was tested in tissue from PCSK9 knockout mice (Supplementary Figure 3C ). After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab).
3
Quantitative Western Blot Analysis
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Frozen LV tissue was rapidly homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodiumdeoxycholate, and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Proteins were quantified using the BCA protein assay (Pierce). A total of 50 ¦Ìg of protein were loaded on a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Filters were blocked and incubated with antibodies against cytochrome oxidase I (Cox I; ThermoFisher Scientific, Waltham, MA, USA, #459600), Cox 15 (abcam, #ab201082), Tfam (Santa Cruz, Santa Cruz, CA, USA, #sc-23588), PGC-1alpha (ThermoFisher, #PA5-72948), alpha AMPK (Cell Signaling, #2532), phospho-AMPK (Cell Signaling, #2535), VDAC1 (Cell Signaling, #4661), alpha 1 AMPK (abcam, Cambridge, UK, #ab32047), alpha 2 AMPK (abcam, #ab3760), Caspase-3 (Cell Signaling, Danvers, MA, USA, #14220 and #9664), and GAPDH (Cell Signaling, #2118). After incubation with a peroxidase-conjugated secondary antibody (1:10.000, Cell Signaling, #7074 and #7076), blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab, Erlangen, Germany).
4
Antibody Immunodetection Protocol
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The following primary antibodies were used in this study: BrdU (Biorad, MCA2060 or Abcam, ab6326, 1:200 dilution); DNA (Progen, AC-30-10, 1:250 dilution); GAPDH (Sigma, G8795 or Abcam, ab8245, 1:10,000 and 1:2000 dilutions, respectively); GRP78 (Santa Cruz Biotech, Sc-13968, 1:1000 dilution); HSP60 (Abcam, ab46798, 1:1000 dilution); LC3B (Sigma, L7543) 1:5000; MTCO-2 (Abcam, ab110258 1:1000 dilution); NDUFB8 (Abcam, ab110242, 1:1000 dilution); AMPK alpha (Cell Signaling, 2532, 1:1000 dilution); phosphoAMPK alpha (Cell Signaling, 2531, 1:1000 dilution); TOM20 (Santa Cruz Biotech or Abcam, Ab186735, 1:4000 and 1:10,000 dilutions, respectively); VCL (Abcam, ab18058, 1:1000 dilution); and proliferating cell nuclear antigen (PCNA) (Mouse, sc-56, 1:8000 dilution).
Secondary Antibodies: Anti-Mouse IgG (H + L), HRP Conjugate (Promega, W4021, 1:4000 dilution); anti-Rabbit IgG (H + L), HRP Conjugate (Promega, W4011 1:4000 dilution); Alexa Fluor®−488 goat- anti-mouse (Invitrogen, A-10684,1:1000 dilution); Alexa Fluor®−568 goat- anti-mouse (Invitrogen, A-11004, 1:1000 dilution); Alexa Fluor® 568 donkey anti-rabbit (Invitrogen, A-10042, 1:1000 dilution); Alexa Fluor®-488 goat- anti-rat (Invitrogen, A-11006, 1:1000 dilution).
Secondary Antibodies: Anti-Mouse IgG (H + L), HRP Conjugate (Promega, W4021, 1:4000 dilution); anti-Rabbit IgG (H + L), HRP Conjugate (Promega, W4011 1:4000 dilution); Alexa Fluor®−488 goat- anti-mouse (Invitrogen, A-10684,1:1000 dilution); Alexa Fluor®−568 goat- anti-mouse (Invitrogen, A-11004, 1:1000 dilution); Alexa Fluor® 568 donkey anti-rabbit (Invitrogen, A-10042, 1:1000 dilution); Alexa Fluor®-488 goat- anti-rat (Invitrogen, A-11006, 1:1000 dilution).
5
Quantifying Akt and AMPK Phosphorylation
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Isolated soleus muscles from 28 weeks old mice were used to determine the levels of Akt and AMPK phosphorylation. Muscles were homogenized in lysis buffer (10% Glycerol, 5% β-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl pH 6.8 and 6 M urea, supplemented with 1% phosphatase inhibitor cocktails 2 and 3 Sigma Aldrich) at a concentration of 10 mg of muscle/ml buffer. Subsequently, 50 µg of protein were separated on a 10% SDS PAG, transferred onto nitrocellulose (Amersham), and probed with the following primary antibodies: phospho-AMPK (Thr172), AMPK alpha, phospho-Akt (ser 473), phopho-Akt (thr 308) and Akt total from Cell Signaling and Desmin (used as housekeeping loading control) from Santa Cruz. The intensity of the immunopositive bands was determined using Image J.
6
Signaling Pathway Antibody Analysis
7
Comprehensive Protein Kinase Inhibitor Protocol
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RPMI-1640 tissue culture medium was obtained from Millipore-Sigma (Burlington, MA, USA) and FBS was obtained from GeminiBio (West Sacramento, CA, USA). All chemicals were obtained from Millipore-Sigma, unless stated otherwise. Complete minitab protease inhibitor (#11836170001), and PhosStop phosphatase inhibitor (#49068450001) were obtained from Millipore-Sigma. Nitrocellulose Western transfer sandwich kit (#1704271) was obtained from Bio-Rad laboratories, (Hong Kong, China). Tyrosine kinase inhibitor osimertinib was obtained from ChemieTek (#CT-A9291) and CDK12 inhibitors AU-15506 (AU-CBB-15506) and AU-16770 (AU-CBB-16770) were provided by Aurigene Discovery Technologies Ltd. (Bengaluru, India). Mouse anti-EGFR mAb was obtained from BD Biosciences (#610017, East Rutherford. NJ, USA). Polyclonal or monoclonal antibodies to pEGFR-Y1068 (#2234), AKT (#4691), ERK (#4370), AMPK-alpha (#5831), ATR (#13934), ATM (#2873), RAD51 (#8875), CHK1 (#2360), CHK2 (#6334), pAKT (#4370, #4370), pERK (#4370), pACC (#11818), and pS6 (#4858) were obtained from Cell Signaling Technology (Danvers, MA, USA). Polyclonal or monoclonal antibodies to CDK12 (ABE1861), RNA Pol II [630849], pSer 2 RNA Pol II (MABE953), and pSer 5 RNA Pol II (MABE954) were obtained from Millipore-Sigma.
8
Antibody Procurement and Western Blot
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Antibodies were purchased from Abcam (S-tag: GR124515-1), Sigma (Flag: F3040), Santa Cruz Biotechnology (Pin1: sc-271441, GAPDH: sc-47724, tubulin: sc-8035 and actin: sc-47778) or Cell Signaling Technology (ACC1: #4190, phospho-ACC1 (Ser79): #11818, ACC2: #8578, AMPK-alpha: #2532, and phosphor-AMPK-alpha (T172): #2535). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui (Tokyo, Japan), SYBR Green from KAPA (Shiga, Japan).
9
Neuronal Protein Extraction and Analysis
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Neurons were plated at a density of 650,000 per well in 12-well plate format. On day 5 after plating, neurons were infected with 3.9E7 TU of each AAV2/6 vector. At day 12, neurons were harvested in lysis buffer containing 0.5% NP40 with protease and phosphatase inhibitors (Roche), and incubated on ice for 10 min. Following cell lysis, protein extracts were centrifuged for 20 min at 14,000 rpm at 4 °C and the supernatant was collected. Protein concentration was evaluated using BCA Protein Kit Assay (Thermofisher Scientific #23225). For detailed description of the protein analysis, refer to Supplementary Methods (Additional file 1 ).
Primary antibodies: AMPK alpha (Cell Signaling #2532) 1:1000; pAMPK alpha (Thr 172) (40H9; Cell Signaling #2535) 1:1000; pACC (Millipore #07–303) 1:1000; Actin (I-19; Santa Cruz #sc-1616) 1:5000; anti-α-syn (Becton Dickinson Biosciences #610787) 1:8000; anti-MAP1LC3B (Lifespan BioSciences) 1:1000. Secondary antibodies: Goat Anti-Rabbit IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401353), Goat Anti-Mouse IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401215).
Primary antibodies: AMPK alpha (Cell Signaling #2532) 1:1000; pAMPK alpha (Thr 172) (40H9; Cell Signaling #2535) 1:1000; pACC (Millipore #07–303) 1:1000; Actin (I-19; Santa Cruz #sc-1616) 1:5000; anti-α-syn (Becton Dickinson Biosciences #610787) 1:8000; anti-MAP1LC3B (Lifespan BioSciences) 1:1000. Secondary antibodies: Goat Anti-Rabbit IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401353), Goat Anti-Mouse IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401215).
10
Western Blot Analysis of Kidney Proteins
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Mouse kidneys were homogenized in lysis buffer containing RIPA buffer and protease inhibitor (#P8340; Sigma Aldrich). Protein concentration was measured using Protein assay dye reagent concentrate (#5000006, Bio-Rad) which is based on the Bradford method. The protein samples were loaded on 4%–20% gradient pre-cast gels (#4568096; Bio-Rad) and transferred to PVDF membranes. The membrane was blocked with 5% BSA, followed by incubation/blotting with primary antibodies of S6 (#2217L; Cell Signaling Technology, Inc.), phospho-S6 (#5364S; Cell Signaling Technology, Inc.), AMPK alpha (#5831S; Cell Signaling Technology, Inc.), phospho-AMPK alpha (#2535S; Cell Signaling Technology, Inc.), LKB1 (#3047S; Cell Signaling Technology, Inc.), phospho-LKB1 (#3482S; Cell Signaling Technology, Inc.), SIRT1 (#9475T; Cell Signaling Technology, Inc.), LC3-I/II (#4108s; Cell Signaling Technology, Inc.), SQSTM1/p62 (#5114; Cell Signaling Technology, Inc.), and GAPDH (#sc-25778; Santa Cruz Biotechnology, Inc.). The working dilution for all the antibodies was 1:1,000. Goat anti-rabbit HRP conjugated secondary antibody was used against the above primary antibodies (#7074S; Cell Signaling Technology, Inc.). The membranes were exposed to ECL reagent (#NEL104001EA; PerkinElmer) and developed using an X-ray film developer. Band density of each blot was quantified using ImageJ software.
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